Abstract:

A procedure was developed to quantify Phytophthora capsici oospores in soil by combining a sieving-centrifugation method and a real-time quantitative polymerase chain reaction (QPCR) assay. Five soil samples representing three different soil textures were infested with oospores of P. capsici to produce 10**1, 10**2, 10**3, 10**4, or 10**5 spores per 10 g of air-dried soil. Each 10-g sample of infested soil was suspended in 400 ml of water and then passed through 106-, 63-, and 38-μm metal sieves. The filtrate was then passed through a 20-μm mesh filter. Materials caught on the filter were washed with water into two 50-ml centrifuge tubes and spun for 4 min (900 × g). The pellet was suspended in 30 ml of 1.6 M sucrose solution and centrifuged for 45 s (190 × g). The supernatant was passed through the 20-μm mesh filter. The sucrose extraction process of oospores was repeated five times to maximize oospore extraction. Materials caught on the 20-μm mesh filter were washed with water into a 50-ml tube and spun for 4 min (900 × g). The pellet was suspended in 1 ml of water, and the number of oospores was determined with a haemocytometer. The relationship between number of oospores recovered from the soil and number of
oospores incorporated into the soil was Ŷ = –0.95 + 1.31X – 0.03X2 (R2 = 0.98), in which Ŷ =
log10 of number of oospores recovered from the soil and X = log10 of number of oospores incorporated into the soil. The oospores were germinated after treatment with 0.1% KMnO4 solution for 10 min to induce germination. On the basis of the detection of ribosomal DNA, a QPCR method for P. capsici oospores was developed. PCR inhibitors were eliminated by extracting oospores from the soil by sieving-centrifugation. DNA was extracted and quantified from P. capsici oospores with suspensions of 10**1, 10**1.5, 10**2, 10**2.5, 10**3, 10**3.5, 10**4, 10**4.5, and 10**5 oospores per ml of water. The relationship between the DNA quantities and number of P. capsici oospores was Ŷ = –3.57 – 0.54X + 0.30X2 (R2 = 0.93), in which Ŷ = log10 (nanogram of P. capsici DNA) and X = log10 (number of oospores). The relationship between the quantity of DNA of P. capsici oospores recovered from the soil and the number of oospores incorporated into the soil was determined by Ŷ = –3.53 – 0.73X + 0.32X2 (R2 = 0.955, P < 0.05), in which Ŷ = log10 (DNA quantity of P. capsici oospores recovered from the soil) and X = log10 (number of P. capsici oospores incorporated into the soil). Utilizing the sieving-centrifugation and QPCR methods, oospores of P. capsici were quantified in soil samples collected from commercial fields.

Introduction:

Among all states, Illinois ranks first in pumpkin production (Babadoost & Islam, 2003). Approximately 20,000 acres of farmland in Illinois are planted to pumpkins annually. More than 90% of the commercial processing pumpkins (Cucurbita moschata) in the United States (US) are produced on approximately 10,000 acres and processed in Illinois (Babadoost & Islam, 2003). Jack-o-lantern pumpkins are produced in more than 10,000 acres throughout Illinois. In addition, approximately 10,000 acres in Illinois are in production of cucumbers, gourds, melons, squashes, watermelons, eggplants, peppers, and tomatoes (Babadoost & Islam, 2003). Peppers are produced in about 1,000 acres. Also, considerable amounts of these vegetables are produced in home gardens throughout the state.

Phytophthora blight of cucurbits, caused by Phytophthora capsici Leonian, is one of the most serious threats to production of cucurbits, pepper, and eggplant worldwide. P. capsici is a soil-borne oomycete that infects more than 50 plant species in 15 families (Erwin and Ribeiro. 1996; Tian and Babadoost, 2004). It can infect plants at any stage of growth, causing seedling damping-off, crown rot, foliage blight, and fruit rot. Phytophthora blight can cause yield losses of up to 100% in cucurbits and pepper fields (Babadoost and Islam, 2003; Hausbeck and Lamour, 2004). At present, there is no long-term sustainable solution for disease management, however, a combination of cultural practices, fungicide application, and genetic resistance can be used to minimize the damages caused by P. capsici to vegetable crops (Babadoost and Islam, 2003; Hausbeck and Lamour, 2004).

P. capsici is a heterothallic organism in which two compatible mating types, designated as A1 and A2, are needed for sexual reproduction. The sexual spore of P. capsici is the oospore, which is the primary source of inoculum and the overwintering propagule in the soil (Erwin and Ribeiro. 1996). A reliable method for quantifying P. capsici oospores in soil is needed to assess survival of the pathogen and the potential for disease development in the field. Methods used for assessing density of P. capsici in soil have been primarily dilution plating and baiting assays but, these methods are time-consuming and the accuracy of the assays is questionable (Erwin and Ribeiro. 1996; Silvar et al., 2005).

Sucrose-centrifugation and sieving is basically a method that separate particles based on their density and size. This method has been used to extract propagules of mycorrhizal fungi (Smith and Skipper, 1979), teliospores of Tilletia species (Babadoost and Mathre, 1998), and nematodes (Jenkins, 1964) from soil.

Silvar et al. (2005) used a polymerase chain reaction (PCR)-based method to detect P. capsici in soil. The protocol they used was for detecting the pathogen only and the method cannot determine quantity of P. capsici oospores in soil. Also, they reported the presence of PCR inhibitory factors in the DNA extracts for molecular detection of plant pathogens in soil (Van de Graaf, 2003). Real-time quantitative polymerase chain reaction (QPCR) is a relatively new molecular technique that has been used to quantify nematodes (Cao et al., 2005), viruses (Delanoy et al., 2003), bacteria (Bach et al., 2003), and fungal plant pathogens (Hayden et al., 2004: Silvar et al., 2005).

The objectives of this study were: 1) to develop a reliable method for quantifying oospores (surviving bodies of P. capsici) in soil (Pavon and Babadoost, 2006a,b,c); 2) to determine survival of P. capsici oospores in soil; and establish effective cropping rotations for management of P. capsici in commercial fields.

Project objectives:

Short-term outcomes of this research were to develop of reliable method for detection and quantification of Phytophthora capsici in soil. Intermediate- and long-term outcomes of this study were to determining survival of P. capsici in soil, establishing effective cropping rotations, reducing use of pesticides, establishing effective strategies for management of one of the most destructive diseases of vegetables, and increasing profitability of vegetable production; thus, establishing sustainable vegetable production in the North Central region.

Any opinions, findings, conclusions, or recommendations expressed in this publication are
those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.

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